Naïve lymphocytes enter different secondary lymphoid organs, primarily lymph nodes, during their constitutive recirculation path through the blood system, to scan for their cognate antigen. To induce an efficient adaptive immune response, T cells must encounter their correspondent antigen which is presented by professional antigen-presenting cells (APCs), primarily dendritic cells, which also migrate towards lymph nodes after efficient antigen capture in the periphery and subsequent maturation. Thus, migration of immune cells towards secondary lymphoid organs represents a prerequisite for the maintenance of the immune system and the induction of an adaptive immune response (5). Generally, migration towards lymph nodes is mediated by the homing chemokine receptor CCR7 and its two ligands, CCL19 and CCL21, which are constitutively expressed within the T cell zone of lymph nodes. CCR7 is expressed on diverse immune cell types, including naïve lymphocytes, mature dendritic cells, Treg cells and a subpopulation of memory T cells known as central memory T cells. Apart from this, CCR7 is found on several cancer cells, supporting metastasis formation within lymph nodes, making CCR7 a prominent object of investigation (93). Similar to all chemokine receptors, CCR7 is a seven-transmembrane-spanning, G-protein coupled receptor (GPCR), which activates the Bordetella pertussis sensitive Gαi
family of G-proteins. Stimulation of CCR7 activates a complex network of intracellular signal transduction pathways, which finally result in a variety of functions, whereof cell polarization and migration is most prominent. Beside its ability to induce directional cell migration, CCR7 has been shown to regulate also other cellular functions, such as cell proliferation and survival (139). Although the implication of CCR7-mediated migration under different immunological aspects is well investigated, the knowledge about precise molecular mechanisms, regulating receptor activity, are currently limited. To shed light on some of these mechanisms, we conducted several studies which supply new aspects on the cellular function of CCR7, in terms of cell signalling and trafficking.
Since function and activity of transmembrane receptors is directly linked to their availability and responsiveness at the plasma membrane, several studies in this work focus on the molecular mechanisms of CCR7 trafficking. Currently, it is well established that only CCL19 affords profound CCR7 internalization, whereas CCL21 stimulation barely induces receptor sequestration, although stimulation with either chemokine elicit G-protein activation and cell migration to a similar extent (97, 98, 100, 126, 127). After initial endocytosis, the receptor is recycled back to the plasma membrane poised to become activated again (98). A previous
study showed that CCR7 internalize via clathrin-coated pits into early endosomes and further colocalizes with transferrin receptor, suggesting a re-expression via recycling endosomes (98). In our present study we confirmed the initial localization of internalized CCR7 within early endosomes and further demonstrated that CCR7 takes the recycling route via the trans-Golgi compartment to reach the plasma membrane (Chapter 2), which was recently also reported for the chemokine receptor CCR5 (171). Surprisingly, our data revealed that the recycling of CCR7 via the trans-Golgi compartment is regulated by the ubiquitylation status of the receptor. Usually, ubiquitylation of GPCRs occurs after ligand treatment and is primarily associated with lysosomal sorting followed by receptor degradation. Regarding chemokine receptors, ubiquitylation was exclusively reported for CXCR4 so far and was also linked to ligand-induced receptor degradation (65, 69, 149). We found a novel role for receptor ubiquitylation in regulating the recycling process of chemokine receptors. We showed that CCR7 is constitutively poly-ubiquitylated on intracellular lysine residues.
Furthermore, we provide clear evidence that this receptor modification is important for correct recycling of internalized CCR7 back to the plasma membrane and does not affect receptor internalization. Confocal microscopy studies revealed that lack of ubiquitylation causes an accumulation of internalized CCR7 after CCL19 stimulation within a perinuclear region, which turned out to be the trans-Golgi compartment. These findings suggest that CCR7 ubiquitylation facilitates the binding or the recruitment of one or more proteins to the internalized receptor within the TGN, which pave the way for CCR7 to the plasma membrane. In this regard, it is interesting to note that GGA proteins function as clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network and are capable to directly bind ubiquitin through their GAT domain (180, 183). Whether the GGA protein family is implicated in the regulation of chemokine receptor trafficking is currently not known and needs further investigations. Apart from receptor recycling, we found an additional function of CCR7 ubiquitylation in the regulation of constitutive receptor trafficking in the absence of chemokine, which is unique for chemokine receptors but has been reported for other GPCRs (164, 165). Although this basal trafficking of CCR7 by receptor ubiquitylation represents an important regulatory mechanism, the main influence on receptor activity is provided by ligand induced receptor internalization, which is associated with receptor desensitization and further cessation of chemokine signalling.
Internalization of CCR7 displays a very interesting field of investigation, as only one of the two known ligands, CCL19, induces distinct receptor endocytosis. Previous studies have shown that CCL19 triggering causes serine/threonine phosphorylation of CCR7 catalyzed by the G-protein coupled receptor kinases GRK3 and GRK6, leading to ß-arrestin2 recruitment, which presumably facilitates receptor internalization (125, 127, 128). In contrast, addition of CCL21 has been shown to activate only GRK6, which also induces CCR7 phosphorylation
and ß-arrestin recruitment, but did not induce receptor endocytosis (128). Structural dissection of CCR7 revealed that distinct serine and threonine clusters within the C-terminal tail of the receptor represent the main CCL19-induced phosphorylation sites (125), suggesting that these regions are important for receptor endocytosis. However the functional consequences of C-terminal CCR7 phosphorylation, in terms of receptor trafficking and cellular function, was not addressed. In order to determine the role of the CCR7 C-terminus we generated three truncation mutants, where the intracellular tail was gradually removed (Chapter 3). All C-terminal serial CCR7 mutants strongly internalized after CCL19 treatment, providing clear evidence that the C-terminal tail is dispensable for CCR7 internalization.
Thus, CCR7 fundamentally differs from several other chemokine receptors including CCR3 (197), CXCR4 (204), CXCR3 (201), CXCR1 and CXCR2 (199), which all showed a strongly impaired internalization upon truncation of the C-terminus. Although CCR7 trafficking was not affected by C-terminal truncation, we identified a novel role for the membrane proximal C terminal residues (aa 335-345) in G-protein activation. Absence of this region as well as mutation of the DRY-motif within the second intracellular loop abrogate the GDP-GTP exchange at the Gα subunit after CCL19 treatment and completely blocks chemokine-mediated cell migration. Collectively, these findings show that G-protein signalling is negligible for CCR7 trafficking, as it was also reported for CXCR3, CXCR4 and CCR5 (46, 205, 207). In an attempt to find the structural components of CCR7 facilitating receptor internalization after ligand binding, we generated a second series of receptor mutants, where all putative phosphorylation sites resided within the intracellular loops (ICLs) of CCR7 were removed (Chapter 4). To this end, we replaced either serine, threonine, or tyrosine clusters within single intracellular loops to investigate whether putative phosphorylation of single ICLs is involved in receptor mediated cellular responses. Similar to our C-terminal truncated receptor mutants, we found that abolishment of Ser/Thr residues within single ICLs did not affect ligand induced receptor internalization. We also generated a CCR7 receptor mutant construct, which lacks all predicted intracellular serine/threonine residues. However, this mutant displayed a severe defect in surface expression, assuming that these residues are required for correct protein folding, membrane insertion or stability at the plasma membrane.
Although mutation of serine and threonine clusters within distinct ICLs did not affect the investigated aspects of chemokine-triggered receptor functions, including receptor trafficking, cell migration and ERK-1/2 activation, we discovered an unexpected function for two tyrosine residues, Y83 and Y85, within the first intracellular loop. Replacement of these tyrosines by phenylalanine leads to a severe defect in G-protein activation after ligand treatment and thus causes a profound inhibition of cell migration. In summary, we discovered by the use of different receptor mutants three distinct regions within the intracellular part of CCR7 which are essential for G-protein activation and thus correct receptor function. Figure 1 depicts a
schematic illustration of CCR7 based on the topology prediction by Swiss-Prot (www.expasy.org/sprot/), where amino acid residues, which are important for G-protein activation are highlighted. Moreover, our data collectively confirmed that G-protein mediated signalling is pivotal for CCR7-triggered chemotaxis but dispensable for ligand-induced receptor internalization and recycling.
Although G-protein signalling plays a crucial role for CCR7-mediated migration, there are potentially also G-protein independent signalling pathways, which can influence CCR7-mediated cellular functions others than receptor trafficking. The prerequisite for G-protein independent signalling is mostly due to posttranslational receptor modifications, such as phosphorylation. As serine/threonine phosphorylation of CCR7 was already investigated to some extend, we assessed the role of tyrosine phosphorylation of CCR7. Our data in chapter 4 clearly demonstrate that CCR7 is indeed tyrosine phosphorylated after stimulation with both ligands in a Src family kinase-dependent and G-protein-independent manner. Ligand induced tyrosine phosphorylation was also reported for the chemokine receptors CCR5, CCR2 and CXCR4 (291-294). For CXCR4, Vila-Coro and co-workers showed that ligand-induced tyrosine phosphorylation resulted in the association and activation of JAK2 and JAK3, enabling the recruitment and tyrosine phosphorylation of several members of the STAT family (294). Moreover, they reported that receptor activation caused activation and recruitment of the tyrosine phosphatase SHP1, however the functional consequences of this receptor association were not further investigated (294). We found that ligand-mediated tyrosine phosphorylation of CCR7 led to the association and activation of the SH2-domain containing tyrosine phosphatase SHP2. Moreover, we have shown that SHP2 is involved in several aspects of CCR7-mediated signalling, including ERK-1/2 activation and cell
Figure 1. CCR7 receptor regions implicated in G-protein activation.
Schematic illustration of CCR7, where regions which are important for ligand-induced G-protein activation are highlighted in green.
migration. Recently, an interesting study revealed that CCL21-mediated receptor stimulation enhanced ERK-1/2 activation upon TCR triggering in mouse lymphocytes, resulting in enhanced T cell proliferation, whereas CCL19 did not display co-stimulatory functions (114).
Based on this finding, we investigated whether CCR7-mediated SHP2 activation participates in chemokine-triggered enhanced T cell proliferation. We found that, in contrast to mouse T cells, both CCR7 ligands have a co-stimulatory effect on human T cell proliferation, whereby SHP2 activity displays a crucial function. Another interesting observation was the biased function for SHP2 in chemokine-induced T cell migration. We identified a severely diminished CCL21-mediated T cell migration upon SHP2 inhibition, whereas CCL19-mediated migration was not affected. This observation was remarkable since up to now there is no evidence that these two ligands exploit different signalling pathways to induce cell migration. A clear biased function for CCL19 and CCL21, as already mentioned, is reported solely in terms of receptor trafficking (128). Given that CCR7-triggering caused recruitment of SHP2 towards the plasma membrane, one could assume that this tyrosine phosphatase operates predominantly at the plasma membrane and thus only hampers CCL21-induced migration significantly. However this hypothesis supposes that CCL19-induced receptor internalization is important for efficient cell migration. Currently, there exists only one publication, which addresses this aspect indirectly. It is reported that lack of ß-arrestin2 and 3 causes diminished CCL19-mediated cell migration, whereas CCL21-triggered migration was not affected (127). Based on this finding one could suggest that receptor internalization, which was shown to depend on ß-arrestin 2, indeed plays a role in cell migration.
Chemokines and their receptors are not isolated entities but instead operate within a complex network of orthogonal signalling pathways arising from the vast number of existing extracellular signalling molecules and target receptors. Nevertheless, there is a high degree of selectivity in crosstalk events, which is further constricted by the protein expression profile of certain cell types. Due to this, we investigated on the influence of TCR signalling on CCR7-mediated cellular responses. In chapter 6 we provide clear evidence that a crosstalk between T cell receptor (TCR) – and CCR7-signalling exists. Previous studies have shown that the TCR also cooperates with the chemokine receptor CXCR4, resulting in a negative crosstalk and feedback modulation of the activity between CXCR4 and the TCR (261). In contrast, we found that short-term activation of freshly isolated human CD3+ T cells with anti-CD3/CD28 positively influences CCR7 signalling, which was reflected in a significantly enhanced chemotactic response towards low concentrations of CCL19 as well as CCL21.
Further investigations revealed that this pro-migratory effect, which became particularly apparent in naïve T cells, was evoked by the different action of the Src family kinases Lck and Fyn in non-activated and short-term activated T cells. Based on our results, we provide a model, depicted in Figure 2 that illustrates TCR/CCR7-signalling-crosstalk, which causes
enhanced migration of (naïve) human T cells towards low concentrations of CCL19 and CCL21.
Currently, it is well defined that intranodal migration of T cells depends on CCR7 (11, 12).
Since the number of antigen-specific naïve T cells is extremely low, it is essential for naïve T cells to scan large areas for their appropriate antigen presenting cell (APC) to initiate an efficient adaptive immune response and thus to stay motile within the initial priming phase, where T cells interact with several APCs for short intervals (260). Our findings provide a first mechanistic insight on how T cells acquire their highly motile phenotype despite opponent stop signals provided by the T cell receptor.
In summary, our results reveal several new insights on the functional and structural regulation of CCR7 which give rise to an ameliorated understanding of ligand-induced receptor trafficking and signalling.
Figure 2. Model of CCR7-mediated migratory response in non-activated and activated human T cells.
In non-activated (naïve) T cells, ligand-mediated activation of CCR7 leads to a PKC-dependent Fyn activation, which causes a negative regulation of Lck by the phosphorylation of its inhibitory tyrosine Y505. TCR engagement prohibits CCR7-mediated Fyn activation either through direct inhibition of Fyn activity or the recruitment of Fyn towards the immunological synapse. This allows direct signalling of CCR7 via Lck. CCR7 triggered activation of Lck results in LAT phosphorylation and permits enhanced cell migration towards CCL19 and CCL21. This mechanism illuminates how naïve T cells maintain their motility in response to CCR7 ligands within lymph nodes during the initial priming phase, despite opponent stop signals provided by the TCR.
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